Heparan sulfate (HS) is required for morphogen signaling during Drosophila pattern formation, but little is known about its physiological importance in mammalian development. To define the developmental role of HS in mammalian species, we conditionally disrupted the HS-polymerizing enzyme EXT1 in the embryonic mouse brain. The EXT1-null brain exhibited patterning defects that are composites of those caused by mutations of multiple HS-binding morphogens. Furthermore, the EXT1-null brain displayed severe guidance errors in major commissural tracts, revealing a pivotal role of HS in midline axon guidance. These findings demonstrate that HS is essential for mammalian brain development.
The response of neuronal growth cones to axon guidance cues depends on the developmental context in which these cues are encountered. We show here that the transmembrane protein semaphorin 5A (Sema5A) is a bifunctional guidance cue exerting both attractive and inhibitory effects on developing axons of the fasciculus retroflexus, a diencephalon fiber tract associated with limbic function. The thrombospondin repeats of Sema5A physically interact with the glycosaminoglycan portion of both chondroitin sulfate proteoglycans (CSPGs) and heparan sulfate proteoglycans (HSPGs). CSPGs function as precisely localized extrinsic cues that convert Sema5A from an attractive to an inhibitory guidance cue. Therefore, glycosaminoglycan bound guidance cues provide a molecular mechanism for CSPG-mediated inhibition of axonal extension. Further, axonal HSPGs are required for Sema5A-mediated attraction, suggesting that HSPGs are components of functional Sema5A receptors. Thus, neuronal responses to Sema5A are proteoglycan dependent and interpreted according to the biological context in which this membrane bound guidance cue is presented.
Materials and methods Targeted recombination of the Ndst1 geneThe thymidine-kinase/neomycin containing targeting vector was constructed by insertion of loxP-sites in intron-sequences surrounding exon 2 (the first coding exon) of Ndst1. The final targeting vector was linearized using SalI before transfection of ES cells. R1 ES cells were grown, transfected and subjected to neomycin G418 selection. Homologous recombinants were identified by Southern blotting and PCR and transfected with a Cre-expressing vector, followed by ganciclovir selection. Four type II recombinants were chosen and injected in C57Bl/6J blastocysts. Two mouse lines, derived from two different ES cell clones, were obtained, and backcrossed into a C57Bl/6 background for more than 10 generations. The primers employed for genotyping were: P1, 5′-CCCAGATGGCGAGACT-GAGG-3′; P2, 5′-CCAGGGCGTCAGGGCCTCCTG-3′; P3, 5′-CATCCTCTGAGGTGACCGC-3′; P4, 5′-GGTACCCGGGGAT-CAATTCG-3′; P5, 5′-CCAGAAGGCTAACACTGTAAAG-3′; P6, 5′-GAAAGTGAAGTCTCTGGGCGG-3′; P7, 5′-GCTTGGATGAT-TTGGTCACACT-3′.Histology and in situ detection of RNA and protein expression Embryos were fixed in 4% paraformaldehyde overnight, dehydrated, embedded in paraffin and sectioned. Sections were stained with Haematoxylin and Eosin for histological analysis. Cartilage and bone were stained with Alcian Blue and Alizarin Red in whole embryos. For whole-mount in situ hybridization a 700 bp riboprobe against the most variable N-terminal region of Ndsts was employed (DIG RNA Labeling Kit, Roche, Mannheim, Germany). Immunohistochemical analysis of Ndst1 expression and western blotting was performed using an anti-Ndst1 antiserum (Grobe and Esko, 2002) followed by detection with goat anti-rabbit HRP-conjugated antibodies (Zymed, San Francisco, USA).Quantitation of apoptosis was performed on paraffin sections of three mutant and three wild-type E15.5 embryos, using the TUNEL Assay Kit (Roche, Mannheim, Germany). BrdU-labelled nuclei were quantified using anti-BrdU antibodies (Zymed) on three mutant and wild-type E15.5 and E17.5 embryos. BrdU (70 μg/g mouse) was injected intraperitoneally and mothers were sacrificed after 1 hour. The number of BrdU-labelled cells relative to nonlabelled cells in the VZ was determined. Two different horizontal levels of the embryonic brains, 250 μm apart, were assessed in each embryo. Eight serial sections per level, each of three wild type and three mutant E17.5 embryos, were analyzed in anterior, posterior, median and lateral forebrain positions.Patched expression was detected using anti-Ptch1 antiserum (Acris Antibodies, Hiddenhausen, Germany) and secondary FITC-labelled goat anti rabbit antibodies (Dianova, Hamburg, Germany) on three mutant and wild-type embryos. Images were taken on a Zeiss Axiophot microscope employing a 10ϫ/0.3, a 20ϫ/0.5 and a 63ϫ/1.25 Zeiss objective, and a Leica DFC280 camera. Leica software was used for image capturing and Photoshop 7 software run on Macintosh computers for the generation of figures. Contrast and brightness were adjusted for whole images ...
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